The three-dimensional (3D) organization of the human genome in the nucleus is important not only for packing 2 meters of DNA inside a 2 micron diameter nucleus, but also for gene regulation and other important biological functions. Taking advantage of the Hi-C technology recently developed to capture chromosome folding and looping throughout the genome within a cell, the proposed research will investigate the relationship between this genome organization and physical disruptions experienced by the cell and DNA. The dramatic physical disruption of a DNA double strand break (DSB) can lead to cancer and other diseases if chromosomal translocations resulting from incorrect break repair disrupt genes or the relationship between genes and regulatory elements. Thus, it is important to understand the factors influencing which translocations are likely to occur after a DSB. Hi-C experiments measuring genome organization in mouse B lymphocytes will be used to determine the extent to which pre-existing proximity of genomic regions correlates with the locations of translocations observed after an induced DSB in the same cells. In cases where a high probability of physical proximity does not explain recurrent translocations, the contribution of other factors to translocation susceptibility will be examined by computationally integrating interaction and translocation data obtained in this research with previously published data on features such as gene expression, protein-DNA interactions, and chromosomal common fragile sites. The influence of the physical properties of the 3D genome structure on the formation of translocations between non-proximal genome regions will then be evaluated by comparing experimentally observed translocations with a biophysical model of the genome derived from principles of polymer physics and experimental Hi-C data. This model will be used to predict how much the physical properties of the genome structure might constrain or enhance the movability of genomic regions and broken ends of DNA in their search for repair partners. These simulations will be followed by direct experimental tests of the deformability of the genome by performing Hi-C experiments on cells subjected to physical forces and constraints. The results of these force experiments will test the biophysical model predictions and enable comparisons between physical deformability and translocation susceptibility. Successfully observing the response of the genome inside cells to physical forces will also provide insight into whether biologically relevant forces in tissues and organs might influence gene expression, cell behavior, and cell fate by direct changes to the chromatin structure. Such insight could contribute to future applications of force in the directed differentiation of cells for tissue engineering purposes. By integrating new experimental data, physical models, and previously published genomic data, this research will contribute to a more complete understanding of the factors involved in the response of the complex 3D genome to physical disruptions in health and disease.